Auto-cascade refrigeration technology has broad application prospects in the field of low-temperature refrigeration. The separation efficiency of refrigerants is one of the main factors affecting system performance. This paper proposes a separation-enhanced auto-cascade refrigeration cycle. An additional cascade heat exchanger and an auxiliary phase separator are introduced before the original cascade heat exchanger. The configuration enhances cooling capacity and cycle performance. The energy, modified exergy, exergo-economic and exergo-environmental performance of the cycle are evaluated through simulation and compared with the basic cycle and previously modified cycle. The results demonstrate that the proposed cycle outperforms both the basic cycle and the modified cycle in both energy and exergy efficiency. The maximum coefficient of performance and exergy efficiency of the separation-enhanced cycle is 44.4% and 44.3% higher than those of the basic cycle, 17.1% and 24.8% higher than those of the modified cycle, respectively. Furthermore, the modified exergy analysis method identifies the exergy destruction caused by the components and zeotropic refrigerant. Actually, the exergy destruction of refrigerant contributes to 39.7% of the overall exergy destruction under typical conditions. Moreover, the exergo-economic analysis indicates a 7.6% reduction in the total cost rate for the proposed cycle compared to the basic cycle and a 2.35% reduction compared to the modified cycle. The exergo-environmental analysis highlights a decrease of 25.4% in overall environmental impact for proposed cycle compared to basic cycle and 12.1% reduction compared to modified cycle. In general, the comparative analysis reveals the improvement potential of the proposed cycle in terms of energy-saving, economic and environmental performance.